The long term objectives of this project are to define the mechanisms that eukaryotic cells employ to replicate damaged DNA. In particular, the genetic control and molecular mechanisms of Rad6-Rad 18 dependent error- free and error-prone bypass replication of UV damaged DNA will be studied in the yeast Saccharomyces cerevisiae. Xeroderma pigmentosum (XP) patients are sensitive to sunlight and they suffer from a high incidence of skin cancers. XP can arise from a defect in nucleotide excision repair, or, in the variant form of XP, from a defect in error-free replication of UV damaged DNA. Error-prone replicative bypass of UV lesions leads to increased mutagenicity in XP cells, the underlying cause of elevated cancer frequency in XP patients. The proposed studies will identify by genetic means the other components of the MMS2-UBC13 dependent error-free postreplicational repair. The biological and functional significance of Rad5 interaction with PCNA will be examined by genetic and biochemical means. The role of Rad5 in DNA unwinding and in D loop formation will be examined, and Rad5-dependent "copy choice" mode of DNA synthesis will be reconstituted with purified proteins. The processivity and fidelity of RAD3O encoded DNA polymerase eta on undamaged DNA will be determined, and genetic and biochemical studies will examine the ability of Pol-eta to bypass a cis-syn T-T dimer and a (6-4) photoproduct. The 3'-> 5' exonuclease activity of Rad 30 will be characterized, and its role in proofreading determined. The role of conserved acidic residues in Rad3O in its DNA polymerase and 3'-> 5' exonuclease activities will be investigated. The Rev3-Rev7 dependent mutagenic translesion synthesis of T-T dimer and of (6-4) photoproduct will be reconstituted with highly purified components, and the role of Rev1 and other proteins such as PCNA and RFC in this process will be analyzed. The DNA replication/damage bypass proteins which become ubiquitinated in response to DNA damage will be identified, and the role of RAD6 and RAD18 in this process will be determined.